The long-term objective of this project is to understand how changes in insulin signaling regulate the actions of amphetamine (AMPH). Dopamine (DA) transporters (DATs), which largely control DA clearance, are targets for psychostimulants such as AMPH and cocaine. By acting on the DAT, AMPH attenuates DA clearance efficiency and induces reverse transport of DA. As a consequence, AMPH increases synaptic DA levels and enhances dopaminergic transmission, with profound effects on behavior. Exciting new experiments suggest that insulin, through phosphatidylinositol 3 kinase (PI3K) signaling, regulates DA clearance by fine-tuning DAT plasma membrane expression. Consistent with these data, in experimentally-induced diabetic rats, where insulin signaling is impaired, DA clearance measured by in vivo chronoamperometry is reduced, as is the ability of AMPH to cause DA efflux. This project will combine biochemistry, biophysics, imaging and in vivo chronoamperometry to elucidate the relationships between changes in PI3K signaling in brain and changes in AMPH-induced increases in extracellular DA. Therefore, the key issues to resolve include how PI3K signaling regulates AMPH-induced DA efflux and to determine, in vivo, whether perturbations in PI3K signaling caused by changes in food intake and disease states such as diabetes regulate the ability of AMPH to increase extracellular DA levels. Interestingly, repeated systemic AMPH, which parallels social intake in humans, overrode the ability of hypoinsulinemia to reduce the effect of acute AMPH. This phenomenon appears to be mediated by D2 receptors. Therefore, we will also evaluate how signaling pathways activated by repeated AMPH exposures (e.g. D2 receptor through ERK1/2 activation) restore the acute actions of AMPH. The proposed studies address the following Specific Aims: 1) To define how PI3K signaling regulates AMPH-induced DA efflux. 2) To demonstrate, in vivo, that hypoinsulinemia or insulin resistance induced by changes in diet, reduce DA clearance and AMPH-induced DA efflux and, ex vivo that these modifications are regulated by DAT trafficking. 3) To determine whether in hypoinsulinemic and insulin resistant rats, repeated administration of AMPH restores AMPH-induced DA efflux by stimulating D2 receptors and consequently ERK1/2. These studies will illuminate pathways that may contribute to the development of psychostimulant abuse. PUBLIC HEALTH RELEVANCE: Stimulant abuse and potentially other dopamine-related pathologies such as schizophrenia and motor disorders (e.g. Parkinson's disease), are a tremendous public health burden. The dopamine transporter, which regulates extracellular brain dopamine, is the major molecular target of several psychoactive drugs, including amphetamine and cocaine. This proposal will analyze how perturbations of insulin signaling induced by diet and diseases states, such as diabetes, regulate dopamine clearance and the ability of amphetamine to increase extracellular brain dopamine, which can lead to addiction. Defining how insulin signaling affects dopamine neurotransmission may help to explain the mechanistic basis of how food intake regulates dopamine neurotransmission and drug abuse.